ABSTRACT Current methods of heart and lung preservation offer extremely limited time between organ donation and transplantation into the recipient. The maximum tolerable cold ischemic times for hearts and lungs are generally considered less than or up to 6 hours, and preferred by cardiac transplant surgeons to be less than 3 hours in clinical practice. In this SBIR project, the goal is to develop a system to preserve donor hearts for a least 24 hours and beyond, allowing unprecedented storage durations of donated hearts. Fully realized, the system will give patients greater access to compatible donor hearts and may substantially reduce the costs associated with urgent and extreme logistics (i.e. allow more time to coordinate the patient, surgeon, and donor heart). At Sylvatica Biotech, the foundation for our approach comes from mechanisms observed in freeze tolerant species combined with principles from bioengineering. The three pillars of innovation in this proposal are isochoric (constant volume) chamber storage, cold storage solution formulations with cryoprotective agents (CPAs) inspired by nature, and advanced multi-thermic machine perfusion. These technologies will be integrated to achieve ice-free, subzero storage of whole hearts. More broadly, the approach can be applied to other organs to prevent ischemic injury and help alleviate key bottlenecks in the development and use of tissue engineered constructs and `humanized' xeno organs. In preliminary work we have demonstrated that a combination of nature-inspired cryoprotective agents (CPAs) and isochoric storage resulted in functional hearts after reaching temperatures as low as -10C. To our knowledge, this is the first demonstration of ventricular function recovery in a whole heart following storage as low as -10C. In this Phase I project, the Sylvatica Biotech team, led by Dr. Michael Taylor and supported by collaborators at UC-Berkeley, the Medical University of South Carolina, and thought leaders in organ preservation, will build on these results. The overall objective is to demonstrate feasibility for the target heart preservation system by storing whole rat hearts at temperatures as low as -20C for at least 24 hours, representing at least a 4x increase over the current standard methods. This will be accomplished in three specific aims: 1) Identify heart-optimized CPA formulations and isochoric temperature control protocols in an in vitro cellular heart model, 2) Develop protocols for preparing whole hearts for isochoric storage using machine perfusion, and 3) Combine the CPA formulations and machine perfusion methods with isochoric storage to prepare whole hearts for subzero storage, store them for at least 24 hours, and recover for functional assessment. Achieving the milestones associated with these aims will show feasibility for the preservation system and justify translating the approach toward human use. In Phase II we will apply these successes to adapt the system to porcine heart and other models of human heart transplant, and work with our established commercialization partners to begin prototyping a clinically-relevant device to prepare and store human donor hearts.